Double Digest Guide
From 2006.igem.org
(Difference between revisions)
(→iGEM Double Digest Guide) |
|||
| Line 1: | Line 1: | ||
| - | = | + | <font size="5">iGEM Double Digest Guide</font><br>by Karmella Haynes, 2006 |
| Line 12: | Line 12: | ||
| - | {|style="width: | + | {|style="width:800px" cellpadding="5" |
| - | |- | + | |- valign="top" |
| - | | '''Enzymes''' || '''Buffer''' | + | | width="150" | '''Enzymes''' |
| - | |- | + | | width="150" | '''Buffer''' |
| + | | '''Temperature''' | ||
| + | | '''Purpose''' | ||
| + | |- valign="top" | ||
| EcoRI, XbaI || Low || 37°C || To create a "Front Vector" | | EcoRI, XbaI || Low || 37°C || To create a "Front Vector" | ||
| - | |- | + | |- valign="top" |
| EcoRI, SpeI || Low || 37°C || To create a "Front Insert" | | EcoRI, SpeI || Low || 37°C || To create a "Front Insert" | ||
| - | |- | + | |- valign="top" |
| SpeI, PstI || Medium || 37°C || To create a "Back Vector" | | SpeI, PstI || Medium || 37°C || To create a "Back Vector" | ||
| - | |- | + | |- valign="top" |
| XbaI, PstI || Low || 37°C || To create a "Back Insert" | | XbaI, PstI || Low || 37°C || To create a "Back Insert" | ||
| - | |- | + | |- valign="top" |
| - | | EcoRI, PstI || Promega® Buffer H || 37°C || To excise entire insert or validate part size | + | | EcoRI, PstI || Promega® Buffer H || 37°C || To excise entire insert or validate part size/ restriction sites |
| + | |- valign="top" | ||
| + | | XbaI, SpeI || Low || 37 º C || To excise entire insert, validate part size/ restriction sites, or clone a PCR with inverted sites | ||
| + | |||
|} | |} | ||
Revision as of 21:58, 7 November 2006
iGEM Double Digest Guide
by Karmella Haynes, 2006
| Standard BioBrick Cloning Sites (Knight) | 5'--GAATTC GCGGCCGC T TCTAGA G ----insert---- T ACTAGT A GCGGCCG CTGCAG-- 3'--CTTAAG CGCCGGCG A AGATCT C -------------- A TGATCA T CGCCGGC GACGTC-- |
| ... | 5'--EcoRI- --NotI-- - -XbaI- - -------------- T -SpeI- - -NotI-- -PstI--- |
| Enzymes | Buffer | Temperature | Purpose |
| EcoRI, XbaI | Low | 37°C | To create a "Front Vector" |
| EcoRI, SpeI | Low | 37°C | To create a "Front Insert" |
| SpeI, PstI | Medium | 37°C | To create a "Back Vector" |
| XbaI, PstI | Low | 37°C | To create a "Back Insert" |
| EcoRI, PstI | Promega® Buffer H | 37°C | To excise entire insert or validate part size/ restriction sites |
| XbaI, SpeI | Low | 37 º C | To excise entire insert, validate part size/ restriction sites, or clone a PCR with inverted sites |
Davidson Buffers [10 mM Tris-HCl pH 7.5, 10 mM MgCl2, 0.1 mg/mL BSA, X mM NaCl]
- 0 (zero), 0 NaCl
- Low, 50 mM NaCl
- Medium, 100 mM NaCl
- High, 150 mM NaCl
Promega® Buffer H [90 mM Tris-HCl pH 7.5, 10 mM MgCl2, 50 mM NaCl]
